Xanthine oxidase assay

XO activity was determined spectrophotometrically by measuring the formation of uric acid from xanthine. The xanthine solution was prepared by initially dissolving xanthine in a minimal volume of NaOH, adjusting pH to 7.5. The XO solution was prepared by diluting to a final concentration of 0.5 U/ml in cold 0.1 M phosphate buffer (pH 7.5). The assay mixture consisted of 200 μL of plant extract solution, 690 μL 0.1 M phosphate buffer (pH 7.5), 60 μL of xanthine solution and 50 μL of XO. The change in absorbance was recorded at 295 nm for 3 min at room temperature.

Allopurinol was used as a standard inhibitor. Xanthine oxidase activity was expressed as percent inhibition of xanthine oxidase, calculated as (A − B)/A × 100, where A is the change in absorbance of the assay without the plant extract, and B is the change in absorbance of the assay with the plant extract. All assays were performed in triplicate.

The IC₅₀ value, a concentration giving 50% inhibition of XO activity, was determined by interpolation of dose-response curves. The mode of inhibition on the enzyme was performed using the Lineweaver–Burk plot. Different concentrations of substrate (20–70 µM) were used for the assay.